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Background: Long noncoding RNAs (lncRNAs) have emerged as novel regulators of macrophage biology and inflammatory cardiovascular diseases. However, studies focused on lncRNAs in human macrophage subtypes, particularly human lncRNAs that are not conserved in rodents, are limited. Methods: Through RNA-sequencing of human monocyte–derived macrophages, we identified suppressor of inflammatory macrophage apoptosis lncRNA ( SIMALR ). Lipopolysaccharide/IFNγ (interferon γ) stimulated human macrophages were treated with SIMALR antisense oligonucleotides and subjected to RNA-sequencing to investigate the function of SIMALR . Western blots, luciferase assay, and RNA immunoprecipitation were performed to validate function and potential mechanism of SIMALR. RNAscope was performed to identify SIMALR expression in human carotid atherosclerotic plaques. Results: RNA-sequencing of human monocyte–derived macrophages identified SIMALR , a human macrophage-specific long intergenic noncoding RNA that is highly induced in lipopolysaccharide/IFNγ–stimulated macrophages. SIMALR knockdown in lipopolysaccharide/IFNγ stimulated THP1 human macrophages induced apoptosis of inflammatory macrophages, as shown by increased protein expression of cleaved PARP (poly[ADP-ribose] polymerase), caspase 9, caspase 3, and Annexin V+. RNA-sequencing of control versus SIMALR knockdown in lipopolysaccharide/IFNγ–stimulated macrophages showed Netrin-1 ( NTN1 ) to be significantly decreased upon SIMALR knockdown. We confirmed that NTN1 knockdown in lipopolysaccharide/IFNγ–stimulated macrophages induced apoptosis. The SIMALR knockdown-induced apoptotic phenotype was rescued by adding recombinant NTN1. NTN1 promoter-luciferase reporter activity was increased in HEK293T (human embryonic kidney 293) cells treated with lentiviral overexpression of SIMALR . NTN1 promoter activity is known to require HIF1α (hypoxia-inducible factor 1 subunit alpha), and our studies suggest that SIMALR may interact with HIF1α to regulate NTN1 transcription, thereby regulating macrophages apoptosis. SIMALR was found to be expressed in macrophages in human carotid atherosclerotic plaques of symptomatic patients. Conclusions: SIMALR is a nonconserved, human macrophage lncRNA expressed in atherosclerosis that suppresses macrophage apoptosis. SIMALR partners with HIF1α (hypoxia-inducible factor 1 subunit alpha) to regulate NTN1, which is a known macrophage survival factor. This work illustrates the importance of interrogating the functions of human lncRNAs and exploring their translational and therapeutic potential in human atherosclerosis.

Background: Cholesterol loaded macrophage foam cells are a prominent feature of atherosclerotic plaques. Single-cell RNA sequencing has identified foam cells as TREM2 (triggering receptor expressed on myeloid cells 2) positive populations with low expression of inflammatory genes, resembling the TREM2 positive microglia of neurodegenerative diseases. Cholesterol loading of macrophages in vitro results in activation of LXR (liver X receptor) transcription factors and suppression of inflammatory genes. Methods: To test the hypothesis that LXRs mediate anti-inflammatory effects in Trem2 expressing atherosclerotic plaque foam cells, we performed RNA profiling on plaque cells from hypercholesterolemic mice with myeloid LXR deficiency. Results: Myeloid LXR deficiency led to a dramatic increase in atherosclerosis with increased monocyte entry, foam cell formation, and plaque inflammation. Bulk cell-RNA profiling of plaque myeloid cells showed prominent upregulation of inflammatory mediators including oxidative, chemokine, and chemotactic genes. Single-cell RNA sequencing revealed increased numbers of foamy TREM2-expressing macrophages; however, these cells had reduced expression of the Trem2 gene expression module, including phagocytic and cholesterol efflux genes, and had switched to a proinflammatory and proliferative phenotype. Expression of Trem2 was suppressed by inflammatory signals but not directly affected by LXR activation in bone marrow-derived macrophages. Conclusions: Our current studies reveal the key role of macrophage LXRs in promoting the Trem2 gene expression program and in suppressing inflammation in foam cells of atherosclerotic plaques.

Long noncoding RNAs (lncRNAs) have emerged as novel regulators of macrophage biology and inflammatory cardiovascular diseases. However, studies focused on lncRNAs in human macrophage subtypes, particularly human lncRNAs that are not conserved in rodents, are limited.Through RNA-sequencing of human monocyte-derived macrophages, we identified suppressor of inflammatory macrophage apoptosis lncRNA (RNA-sequencing of human monocyte-derived macrophages identified

Background: Hypertrophic cardiomyopathy often causes major adverse cardiovascular events (MACE), for example, arrhythmias, stroke, heart failure, and sudden cardiac death. Currently, there are no models available to predict MACE. Furthermore, it remains unclear which signaling pathways mediate MACE. Therefore, we aimed to prospectively determine protein biomarkers that predict MACE in hypertrophic cardiomyopathy and to identify signaling pathways differentially regulated in patients who subsequently develop MACE. Methods: In this multi-centre prospective cohort study of patients with hypertrophic cardiomyopathy, we conducted plasma proteomics profiling of 4979 proteins upon enrollment. We developed a proteomics-based model to predict MACE using data from one institution (training set). We tested the predictive ability in independent samples from the other institution (test set) and performed time-to-event analysis. Additionally, we executed pathway analysis of predictive proteins using a false discovery rate threshold of Results: The study included 245 patients (n=174 in the training set and n=71 in the test set). Using the proteomics-based model to predict MACE derived from the training set, the area under the receiver-operating-characteristic curve was 0.81 (95% CI, 0.68–0.93) in the test set. In the test set, the high-risk group determined by the proteomics-based predictive model had a significantly higher rate of developing MACE (hazard ratio, 13.6 [95% CI, 1.7–107]; P =0.01). The Ras -MAPK (mitogen-activated protein kinase) pathway was upregulated in patients who subsequently developed MACE (false discovery rate-7 ). Pathways involved in inflammation and fibrosis—for example, the TGF (transforming growth factor)-β pathway—were also upregulated. Conclusions: This study serves as the first to demonstrate the ability of proteomics profiling to predict MACE in hypertrophic cardiomyopathy, exhibiting both novel (eg, Ras -MAPK) and known (eg, TGF-β) pathways differentially regulated in patients who subsequently experience MACE.

Background: Long noncoding RNAs (lncRNAs) are important regulators of biological processes involved in vascular tissue homeostasis and disease development. The present study assessed the functional contribution of the lncRNA myocardial infarction-associated transcript ( MIAT ) to atherosclerosis and carotid artery disease. Methods: We profiled differences in RNA transcript expression in patients with advanced carotid artery atherosclerotic lesions from the Biobank of Karolinska Endarterectomies. The lncRNA MIAT was identified as the most upregulated noncoding RNA transcript in carotid plaques compared with nonatherosclerotic control arteries, which was confirmed by quantitative real-time polymerase chain reaction and in situ hybridization. Results: Experimental knockdown of MIAT , using site-specific antisense oligonucleotides (LNA-GapmeRs) not only markedly decreased proliferation and migration rates of cultured human carotid artery smooth muscle cells (SMCs) but also increased their apoptosis. MIAT mechanistically regulated SMC proliferation through the EGR1 (Early Growth Response 1)-ELK1 (ETS Transcription Factor ELK1)-ERK (Extracellular Signal-Regulated Kinase) pathway. MIAT is further involved in SMC phenotypic transition to proinflammatory macrophage-like cells through binding to the promoter region of KLF4 and enhancing its transcription. Studies using Miat –/– and Miat –/– ApoE –/– mice, and Yucatan LDLR –/– mini-pigs, as well, confirmed the regulatory role of this lncRNA in SMC de- and transdifferentiation and advanced atherosclerotic lesion formation. Conclusions: The lncRNA MIAT is a novel regulator of cellular processes in advanced atherosclerosis that controls proliferation, apoptosis, and phenotypic transition of SMCs, and the proinflammatory properties of macrophages, as well.

Septal reduction therapy (SRT) -i.e. septal myectomy and alcohol septal ablation-has been performed to treat medically refractory hypertrophic cardiomyopathy (HCM) for decades. However, it is largely unknown whether SRT prevents HCM-related cardiovascular events or death. The objective was to examine the effects of SRT on acute cardiovascular events and all-cause mortality in HCM. We performed a propensity score (PS) -matched study using databases that capture all hospitalizations and outpatient visits in New York state. We identified patients with HCM who underwent SRT between 2007 and 2014 (i.e. the SRT group) and those who had never had SRT but had at least one hospitalization for HCM during the same period (i.e. the control group). We performed PS matching at a 1:1 ratio. The primary outcome was a composite of acute cardiovascular events and all-cause mortality during 0-180 days and 181-360 days. The secondary outcome was 180- and 360-day all-cause mortality. We included 846 patients with HCM (423 PS-matched pairs). Patients who underwent SRT had a lower risk of the primary outcome event (0-180 days: odds ratio [OR], 0.54; 95% confidence intervals (CI), 0.37-0.80; P = 0.002 and 181-360 days: OR, 0.33; 95% CI, 0.22-0.51; P < 0.0001). Furthermore, the risk of all-cause mortality was lower at 180 days (OR, 0.37; 95% CI, 0.22-0.63; P = 0.0003) and 360 days post-SRT (OR, 0.32; 95% CI, 0.20-0.51; P < 0.0001). In conclusion, our PS-matched study using population-based datasets demonstrated that SRT was associated with a reduced risk of a composite of acute cardiovascular events and all-cause mortality in HCM during the first post-SRT year.

Background Obesity is an established risk factor for severe COVID-19 outcomes. The mechanistic underpinnings of this association are not well-understood. Objective To evaluate the mediating role of systemic inflammation in obesity-associated COVID-19 outcomes. Methods This hospital-based, observational study included 3828 SARS-CoV-2-infected patients who were hospitalized February to May 2020 at Massachusetts General Hospital (MGH) or Columbia University Irving Medical Center/New York Presbyterian Hospital (CUIMC/NYP). We use mediation analysis to evaluate whether peak inflammatory biomarkers (C-reactive protein [CRP], erythrocyte sedimentation rate [ESR], D-dimer, ferritin, white blood cell count and interleukin-6) are in the causal pathway between obesity (BMI ≥ 30) and mechanical ventilation or death within 28 days of presentation to care. Results In the MGH cohort (n = 1202), obesity was associated with greater likelihood of ventilation or death (OR = 1.73; 95% CI = [1.25, 2.41]; P = 0.001) and higher peak CRP (P Conclusion Findings support systemic inflammatory pathways in obesity-associated severe COVID-19 disease, particularly in patients

BACKGROUNDSmooth muscle cells (SMCs) substantially contribute to the development of atherosclerosis through a process called “phenotypic switching.” Our previous work identified an SMC-derived intermediate cell type, termed “SEM” cells, which plays a crucial role in SMC transition to other cell types and in lesion development. Activation of retinoic acid (RA) signaling by all-trans retinoic acid (ATRA) attenuates atherosclerosis in mice coincident with suppression of SEM cell formation. However, the effect of RA signaling on advanced disease and the underlying molecular mechanisms by which RA modulates SMC transition to SEM cells are largely unknown.METHODSWe applied SMC lineage tracing atheroprone mice and biochemistry and cell and molecular biology techniques (e.g., RNA sequencing, quantitative reverse transcription PCR, co-immunoprecipitation, and chromatin immunoprecipitation-quantitative PCR) to reveal the regulatory mechanisms of RA signaling in SMC transition to SEM cells.RESULTSActivation of RA signaling with ATRA in established atherosclerosis significantly reduced SEM cells and lesion size while increasing fibrous cap thickness. Mechanistically, retinoic acid receptor alpha (RARα) directly targets the promoters ofLy6aandLy6c1in mouse SMCs, and activation of RA signaling recruits EZH2 to the regulatory elements triggering local H3K27me3. Distinct from a molecular model that reported for RA recruitment of HDAC1 during embryogenesis, RARα/EZH2 complex recruits SIRT1 and SIRT6, rather than classical HDACs, to the regulatory regions of key SEM cell marker genes. This subsequently reduces multiple acetylated histone modifications (e.g., H3K27ac, H3K4ac, H3K9ac, H3K14ac, H3K56ac) with recruitment of the transcription corepressor, NCOR1, to repress downstream SEM cell marker genes.CONCLUSIONSOur findings provide novel mechanistic insights into RA modulating SMC phenotypic switching in atherosclerosis, suggesting molecular targets for preventive and therapeutic interventions for atherosclerosis and its clinical complications.

CITE-seq, a single-cell multi-omics technology that measures RNA and protein expression simultaneously in single cells, has been widely applied in biomedical research, especially in immune related disorders and other diseases such as influenza and COVID-19. Despite the proliferation of CITE-seq, it is still costly to generate such data. Although data integration can increase information content, this raises computational challenges. First, combining multiple datasets is prone to batch effects that need to be addressed. Secondly, it is difficult to combine multiple CITE-seq datasets because the protein panels in different datasets may only partially overlap. Integrating multiple CITE-seq and single-cell RNA-seq (scRNA-seq) datasets is important because this allows the utilization of as many data as possible to uncover cell population heterogeneity. To overcome these challenges, we present sciPENN, a multi-use deep learning approach that supports CITE-seq and scRNA-seq data integration, protein expression prediction for scRNA-seq, protein expression imputation for CITE-seq, quantification of prediction and imputation uncertainty, and cell type label transfer from CITE-seq to scRNA-seq. Comprehensive evaluations spanning multiple datasets demonstrate that sciPENN outperforms other current state-of-the-art methods.

Hundreds of interventional clinical trials have been launched in the United States to identify effective treatment strategies for combating the coronavirus disease 2019 (COVID-19) pandemic. However, to date, only a small fraction of these trials have completed enrollment, delaying the scientific investigation of COVID-19 and its treatment options. This study presents novel metrics to examine the geographic alignment between COVID-19 hotspots and interventional clinical trial sites and evaluate trial access over time during the evolving pandemic. Using temporal COVID-19 case data from USAFacts.org and trial data from ClinicalTrials.gov, U.S. counties were categorized based on their numbers of cases and trials. Our analysis suggests that alignment and access have worsened as the pandemic shifted over time. We recommend strategies and metrics to evaluate the alignment between cases and trials. Future studies are warranted to investigate the impact of the misalignment of cases and clinical trial sites on clinical trial recruitment.

Age is the dominant risk factor for infectious diseases, but the mechanisms linking the two are incompletely understood1,2. Age-related mosaic chromosomal alterations (mCAs) detected from blood-derived DNA genotyping, are structural somatic variants associated with aberrant leukocyte cell counts, hematological malignancy, and mortality3-11. Whether mCAs represent independent risk factors for infection is unknown. Here we use genome-wide genotyping of blood DNA to show that mCAs predispose to diverse infectious diseases. We analyzed mCAs from 767,891 individuals without hematological cancer at DNA acquisition across four countries. Expanded mCA (cell fraction10%) prevalence approached 4% by 60 years of age and was associated with diverse incident infections, including sepsis, pneumonia, and coronavirus disease 2019 (COVID-19) hospitalization. A genome-wide association study of expanded mCAs identified 63 significant loci. Germline genetic alleles associated with expanded mCAs were enriched at transcriptional regulatory sites for immune cells. Our results link mCAs with impaired immunity and predisposition to infections. Furthermore, these findings may also have important implications for the ongoing COVID-19 pandemic, particularly in prioritizing individual preventive strategies and evaluating immunization responses.

Clonal haematopoiesis, which is highly prevalent in older individuals, arises from somatic mutations that endow a proliferative advantage to haematopoietic cells. Clonal haematopoiesis increases the risk of myocardial infarction and stroke independently of traditional risk factors1. Among the common genetic variants that give rise to clonal haematopoiesis, the JAK2V617F (JAK2VF) mutation, which increases JAK–STAT signalling, occurs at a younger age and imparts the strongest risk of premature coronary heart disease1,2. Here we show increased proliferation of macrophages and prominent formation of necrotic cores in atherosclerotic lesions in mice that express Jak2VF selectively in macrophages, and in chimeric mice that model clonal haematopoiesis. Deletion of the essential inflammasome components caspase 1 and 11, or of the pyroptosis executioner gasdermin D, reversed these adverse changes. Jak2VF lesions showed increased expression of AIM2, oxidative DNA damage and DNA replication stress, and Aim2 deficiency reduced atherosclerosis. Single-cell RNA sequencing analysis of Jak2VF lesions revealed a landscape that was enriched for inflammatory myeloid cells, which were suppressed by deletion of Gsdmd. Inhibition of the inflammasome product interleukin-1β reduced macrophage proliferation and necrotic formation while increasing the thickness of fibrous caps, indicating that it stabilized plaques. Our findings suggest that increased proliferation and glycolytic metabolism in Jak2VF macrophages lead to DNA replication stress and activation of the AIM2 inflammasome, thereby aggravating atherosclerosis. Precise application of therapies that target interleukin-1β or specific inflammasomes according to clonal haematopoiesis status could substantially reduce cardiovascular risk. Accelerated atherosclerosis in a mouse model of clonal haematopoiesis is prevented by genetic interruption of AIM2 inflammasome activation or by inhibition of interleukin-1β.

Single Cell RNA Sequencing (scRNA-seq) has proven to be a powerful technique to uncover heterogeneity from otherwise homogeneous tissues. Multiple studies have utilized scRNA-seq to examine the composition of atherosclerotic plaques in both humans and mice. However, most of these studies occurred in advanced atherosclerosis. We sought to expand this knowledge base by performing single-cell RNA sequencing at early atherosclerosis in mice. We crossed the Ldlr-/- hyperlipidemic mouse model to a smooth muscle cell (SMC) lineage tracing mouse (ROSA26:LSL-ZsGreen; MyH11-CreERT2). When treated with tamoxifen, SMCs expressing Myh11 are labeled green. At seven weeks of age, we treated these mice with five IP injections of 40mg/kg of tamoxifen. After one week of rest, we initiated the mice on a western diet. We dissected out the whole aorta at the time of harvest and digested the aorta for one hour with a cocktail of DNase, Hyaluronidase, and Liberase. Subsequently, we FACS sorted out both ZsGreen negative and ZsGreen positive cells and performed single-cell RNA seq using the 10x Genomics pipeline. We performed scRNA-seq with pools of three mice at four time points: after 1, 2, 3, and 4 weeks of western diet feeding. Clustering with CarDEC revealed three SMC populations. At four weeks of western diet, we observed an emergence of a SMC cluster characterized by increased expression of Vcam1, Dcn, Mgp, and Egr1. As expected, Gene Ontology analysis of the top 20 differentially expressed genes revealed this population to contain reduced markers of SMC differentiation. In addition, this population had reduced expression of Pura, a protein known to influence serum response factor and regulate smooth muscle actin expression. In summary, our studies indicate changes within SMCs, and the emergence of a dedifferentiated smooth muscle cell population after four weeks of western diet feeding. This study highlights that remodeling of the vasculature occurs even with short durations of hyperlipidemia. Further ongoing studies include expanding time points to longer durations of western diet and elucidating master regulators of this population with Virtual Inference of Protein activity by Enriched Regulon (VIPER).

Patients with JAK2 V617F (JAK2 VF ) clonal hematopoiesis (CH) have increased JAK/STATsignaling that imparts an increased risk of premature coronary heart disease. We have shown that mice with Jak2 VF mutations modeling CH have increased AIM2 inflammasome activation that promotes atherosclerosis. Genetic inhibition of the inflammasome through deletion of Caspase1/11 or Gasdermin D in hematopoietic stem and progenitor cells (HSPC) harboring Jak2 VF mutations increased fibrous caps thickness. Therefore, we hypothesized that inhibition of IL-1β derived from Jak2 VF macrophages promotes smooth muscle cell (SMC) reprograming and accumulation in fibrous caps. To test this hypothesis, we transplanted bone marrow (BM) with Jak2 VF expression in all HSPCs into Ldlr -/- mice and then administered the IL-1 receptor antagonist Anakinra. After 12 weeks of Western type diet (WTD) feeding, mice treated with Anakinra had thicker fibrous caps and increased ACTA2 + SMCs within the cap region. In atheromas scRNA-Seq has identified SMC-derived populations including modified SMCs and fibroblasts which are enriched for Prg4 . Consistently, Anakinra treatment increased Prg4 -expressing cells within plaques as shown by RNA scope in situ hybridization. We then modeled Jak2 VF CH by transplanting mixtures of 20% Jak2 VF BM (or 20% littermate control BM) with 80% WT BM into Ldlr -/- mice followed by administration of antibodies to IL-1β. After WTD feeding, IL-1β antibody treatment led to increased cap thickness specifically in mice modeling Jak2 VF CH. To identify how IL-1β may alter cap thickness we isolated CD11b Negative cells from aortas and conducted bulk-cell RNA-Seq. Gene ontology analysis revealed that IL-1β antagonism specifically in Jak2 VF CH mice led to the significant enrichment of 6 biological processes all of which were related to biosynthetic processes including translation, peptide biosynthetic processes, peptide metabolism and amide biosynthesis. These changes were not seen in control mice indicating that inhibition of IL-1β derived from Jak2 VF hematopoietic cells leads to increased translation and biosynthetic processes in non-myeloid cells potentially supporting SMC differentiation and migration and resulting in increased fibrous cap formation.

Background: Dysfunctional adipose tissue (AT), characterized by increased inflammation and immune cell recruitment, plays a central role in the development of T2DM and atherosclerotic CVDs. LincRNAs are important emerging regulators of cellular functions. Methods: RNA-seq of human AT during low-dose endotoxemia, identified novel lincRNAs regulated by inflammation. LincADAIN was identified as 1)specifically expressed in AT 2)reduced in both obesity-induced chronic and LPS-induced acute AT inflammation (-77% and -53%, pin vitro , prior to injection. After 16 weeks, implants are extracted and analyzed for adipocyte morphology, immune cell infiltration and gene expression changes. Biotinylated lincRNA pulldown assay was used to identify interacting proteins by Mass Spectrometry (MS) and hits validated via RNA IP assays. Cytokines from LPS stimulated adipocytes after Linc-ADAIN KD were measured via cytokine array. Results: Human LincADAIN expression negatively correlates to BMI in obese AT (p Conclusion: Linc-ADAIN is a novel lincRNA that can regulate AT inflammation potentially through modulation of RNA translation via RBPs. Our mouse model provides a foundation to uncover Linc-ADAIN’s role in AT inflammation in vivo and to study other non-conserved human lincRNAs. Functional investigations of lincRNAs, such as these are warranted, as recent genomic studies suggest that lincRNAs are likely the causal element driving human disease at some GWAS loci.

Background: Cardiovascular diseases, including atherosclerosis, are the major cause of death in western societies, still molecular mechanisms of plaque destabilization remain unclear. Long non-coding RNAs (lncRNAs) are one example of novel molecular modulators, as their expression is highly cell-type specific. Methods: We utilized combined total (bulk) RNA and single cell (sc) RNA to study the transcriptome of advanced carotid artery lesions from patients undergoing carotid endarterectomy in our vascular surgery clinic. Additionally, we performed hybridization-based RNA in situ sequencing (HybRISS) to indicate where cluster-defining genes are located within the plaques. Results: In this current study, four sequencing datasets were investigated (total RNA from early vs. late lesions from the same individual patient and unstable vs. stable lesions from individual patients; two separate scRNA-seq datasets).16 lncRNAs were cross-referenced between all four datasets. All of these lncRNAs presented a cell-type specific expression pattern, with 11 lncRNAs being significantly enriched in different smooth muscle cell (SMC) clusters. We found all newly identified lncRNAs conserved in our scRNA-seq datasets of genetically mutated ( LDLR-/- ) Yucatan mini-pigs and the inducible carotid artery plaque rupture mice ( ApoE-/- ). The cluster-defining genes from the human scRNA-seq data were then located in human carotid artery tissue sections using the HybRISS method to unravel their distinct location within these plaques. Discussion: Taken together, our datasets, methods and different animal-models demonstrate that combining bulk with scRNA-seq data and spatially resolved sequencing methods are powerful tools to identify and characterize novel lncRNAs being expressed by a certain cell-type in the disease progression.

BackgroundHuman heart failure is characterized by global alterations in the myocardial DNA methylation profile, yet little is known about epigenetic regulation of non-coding transcripts and potential reversibility of DNA methylation with left ventricular assist device (LVAD) support.MethodGenome-wide mapping of myocardial DNA methylation was performed in 36 patients with end-stage heart failure at the time of LVAD implant, 8 patients at the time of LVAD explant, and 7 non-failing controls using high-density bead array platform. Transcriptomic and functional studies were performed in human induced pluripotent stem cell derived cardiomyocytes (iPSCs).ResultsEtiology-specific analysis revealed 2079 differentially methylated positions (DMPs) in ischemic cardiomyopathy (ICM) and 261 DMPs in non-ischemic cardiomyopathy (NICM). 192 DMPs were common to ICM and NICM. Analysis of paired samples before and after LVAD support demonstrated reverse methylation of only 3.2% of HF-specific DMPs. Methylation-expression correlation analysis yielded several protein-coding genes that are hypomethylated and upregulated (HTRA1, FAM65A, FBXO16, EFCAB13, AKAP13, RPTOR) or hypermethylated and downregulated (TBX3) in ICM and NICM patients. A novel cardiac-specific super-enhancer lncRNA (LINC00881) is hypermethylated and downregulated in the failing human heart.LINC00881is an upstream regulator of sarcomere and calcium channel gene expression includingMYH6, CACNA1C, andRYR2. LINC00881knockdown significantly reduced peak calcium amplitude in the beating human iPSCs.ConclusionsFailing human heart exhibits etiology-specific changes in DNA methylation including coding and non-coding regions, which are minimally reversible with mechanical unloading. Epigenetic reprogramming may be necessary to achieve transcriptional normalization and sustained clinical recovery from heart failure.

Background: Smooth muscle cells (SMCs) play significant roles in atherosclerosis via phenotypic switching, a pathological process in which SMC dedifferentiation, migration, and transdifferentiation into other cell types. Yet how SMCs contribute to the pathophysiology of atherosclerosis remains elusive. Methods: To reveal the trajectories of SMC transdifferentiation during atherosclerosis and to identify molecular targets for disease therapy, we combined SMC fate mapping and single-cell RNA sequencing of both mouse and human atherosclerotic plaques. We also performed cell biology experiments on isolated SMC-derived cells, conducted integrative human genomics, and used pharmacological studies targeting SMC-derived cells both in vivo and in vitro. Results: We found that SMCs transitioned to an intermediate cell state during atherosclerosis, which was also found in human atherosclerotic plaques of carotid and coronary arteries. SMC-derived intermediate cells, termed “SEM” cells (stem cell, endothelial cell, monocyte), were multipotent and could differentiate into macrophage-like and fibrochondrocyte-like cells, as well as return toward the SMC phenotype. Retinoic acid (RA) signaling was identified as a regulator of SMC to SEM cell transition, and RA signaling was dysregulated in symptomatic human atherosclerosis. Human genomics revealed enrichment of genome-wide association study signals for coronary artery disease in RA signaling target gene loci and correlation between coronary artery disease risk alleles and repressed expression of these genes. Activation of RA signaling by all-trans RA, an anticancer drug for acute promyelocytic leukemia, blocked SMC transition to SEM cells, reduced atherosclerotic burden, and promoted fibrous cap stability. Conclusions: Integration of cell-specific fate mapping, single-cell genomics, and human genetics adds novel insights into the complexity of SMC biology and reveals regulatory pathways for therapeutic targeting of SMC transitions in atherosclerotic cardiovascular disease.

Understanding the clinical risk factors for COVID-19 disease severity and outcomes requires a combination of data from electronic health records and patient reports. To facilitate the collection of patient-reported data, as well as accelerate and standardize the collection of data about host factors, we have constructed a COVID-19 survey. This survey is freely available to the scientific community to send electronically for patients to complete online. This patient survey is designed to be comprehensive, yet not overly burdensome, to gather data useful for a range of clinical investigations, and to accommodate a wide variety of implementation settings including at a COVID-19 testing site, at home during infection or after recovery, and/or for individuals while they are hospitalized. A widely adopted standardized survey that can be implemented online with minimal resources can serve as a critical tool for combining and comparing data across studies to improve our understanding of COVID-19 disease.

It is unclear how hypertrophic cardiomyopathy (HCM) affects cardiac metabolic pathways at rest and with exercise. This case–control study compared 15 cases with HCM to 2 control groups without HCM. Metabolomic profiling of 210 metabolites was carried out at rest and at peak exercise. The 50 most discriminant metabolites differentially regulated during exercise were selected using partial least squares discriminant analysis. Pathway enrichment analysis was also performed. At rest, no significant difference was observed in metabolomic profiling of HCM cases as compared to controls. By contrast, there were significant differences in metabolomic profiling in response to exercise (p

This review focuses on the human genetics, epidemiology, and molecular pathophysiology of sex differences in central obesity, adipose distribution, and related cardiometabolic disorders. Distribution of fat is important for cardiometabolic health, with peripheral fat depots having a protective effect and central visceral fat depots conferring a detrimental effect on health. There are important sex differences in fat distribution that are masked when studying body mass index as a measure of obesity. From epidemiological, murine, and in vitro studies, several mechanisms have been proposed to explain the sex differences in adipose distribution, including sex hormonal effects, cell-intrinsic properties, and the microenvironment in fat depots. More recently, human genetics have revealed hundreds of loci for central obesity providing disruptive opportunities for mechanistic discoveries and clinical translation. A striking feature is that over one-third of these loci have reproducible but poorly understood sexual dimorphic associations with central obesity, most having stronger effects in women. Understanding the genetic and molecular mechanisms of adipose distribution and its sexual dimorphism in humans provides a unique opportunity to promote the use of precision medicine for early identification of at-risk individuals, and the development of novel therapeutic strategies for central obesity and related cardiometabolic disorders.

Many complex disease risk loci map to intergenic regions containing long intergenic noncoding RNAs (lincRNAs). The majority of these is not conserved outside humans, raising the question whether genetically regulated expression of non-conserved and conserved lincRNAs has similar rates of association with complex traits. Here we leveraged data from the Genotype-Tissue Expression (GTEx) project and multiple public genome-wide association study (GWAS) resources. Using an established transcriptome-wide association study (TWAS) tool, FUSION, we interrogated the associations between cis-regulated expression of lincRNAs and multiple cardiometabolic traits. We found that cis-regulated expression of non-conserved lincRNAs had a strikingly similar trend of association with complex cardiometabolic traits as conserved lincRNAs. This finding challenges the conventional notion of conservation that has led to prioritization of conserved loci for functional studies and calls attention to the need to develop comprehensive strategies to study the large number of non-conserved human lincRNAs that may contribute to human disease.

Objective: LIPA (lysosomal acid lipase) mediates cholesteryl ester hydrolysis, and patients with rare loss-of-function mutations develop hypercholesterolemia and severe disease. Genome-wide association studies of coronary artery disease have identified several tightly linked, common intronic risk variants in LIPA which unexpectedly associate with increased mRNA expression. However, an exonic variant (rs1051338 resulting in T16P) in linkage with intronic variants lies in the signal peptide region and putatively disrupts trafficking. We sought to functionally investigate the net impact of this locus on LIPA and whether rs1051338 could disrupt LIPA processing and function to explain coronary artery disease risk. Approach and Results: In monocytes isolated from a large cohort of healthy individuals, we demonstrate both exonic and intronic risk variants are associated with increased LIPA enzyme activity coincident with the increased transcript levels. To functionally isolate the impact of rs1051338, we studied several in vitro overexpression systems and consistently observed no differences in LIPA expression, processing, activity, or secretion. Further, we characterized a second common exonic coding variant (rs1051339), which is predicted to alter LIPA signal peptide cleavage similarly to rs1051338, yet is not linked to intronic variants. rs1051339 also does not impact LIPA function in vitro and confers no coronary artery disease risk. Conclusions: Our findings show that common LIPA exonic variants in the signal peptide are of minimal functional significance and suggest coronary artery disease risk is instead associated with increased LIPA function linked to intronic variants. Understanding the mechanisms and cell-specific contexts of LIPA function in the plaque is necessary to understand its association with cardiovascular risk.

ObjectiveHypertrophic cardiomyopathy (HCM) is a heterogeneous disease, likely encompassing several subtypes of disease with distinct biological mechanisms (ie, molecular subtypes). Current models based solely on clinical data have yielded limited accuracy in predicting the risk of major adverse cardiovascular events (MACE). Our aim in this study was to derive molecular subtypes in our multicentre prospective cohort of patients with HCM using proteomics profiling and to examine their longitudinal associations with MACE.MethodsWe applied unsupervised machine learning methods to plasma proteomics profiling data of 1681 proteins from 258 patients with HCM who were prospectively followed for a median of 2.8 years. The primary outcome was MACE, defined as a composite of arrhythmia, heart failure, stroke and sudden cardiac death.ResultsWe identified four molecular subtypes of HCM. Time-to-event analysis revealed significant differences in MACE-free survival among the four molecular subtypes (plogrank=0.007). Compared with the reference group with the lowest risk of MACE (molecular subtype A), patients in molecular subtype D had a higher risk of subsequently developing MACE, with an HR of 3.41 (95% CI 1.54 to 7.55, p=0.003). Pathway analysis of proteins differentially regulated in molecular subtype D demonstrated an upregulation of the Ras/mitogen-activated protein kinase and associated pathways, as well as pathways related to inflammation and fibrosis (eg, transforming growth factor-β pathway).ConclusionsOur prospective plasma proteomics study not only exhibited the presence of HCM molecular subtypes but also identified pathobiological mechanisms associated with a distinct high-risk subtype of HCM.

Background and aims The atherosclerotic plaque microenvironment is highly complex, and selective agents that modulate plaque stability are not yet available. We sought to develop a scRNA-seq analysis workflow to investigate this environment and uncover potential therapeutic approaches. We designed a user-friendly, reproducible workflow that will be applicable to other disease-specific scRNA-seq datasets. Methods Here we incorporated automated cell labeling, pseudotemporal ordering, ligand-receptor evaluation, and drug-gene interaction analysis into a ready-to-deploy workflow. We applied this pipeline to further investigate a previously published human coronary single-cell dataset by Wirka et al. Notably, we developed an interactive web application to enable further exploration and analysis of this and other cardiovascular single-cell datasets. Results We revealed distinct derivations of fibroblast-like cells from smooth muscle cells (SMCs), and showed the key changes in gene expression along their de-differentiation path. We highlighted several key ligand-receptor interactions within the atherosclerotic environment through functional expression profiling and revealed several avenues for future pharmacological development for precision medicine. Further, our interactive web application, PlaqView ( www.plaqview.com ), allows lay scientists to explore this and other datasets and compare scRNA-seq tools without prior coding knowledge. Conclusions This publicly available workflow and application will allow for more systematic and user-friendly analysis of scRNA datasets in other disease and developmental systems. Our analysis pipeline provides many hypothesis-generating tools to unravel the etiology of coronary artery disease. We also highlight potential mechanisms for several drugs in the atherosclerotic cellular environment. Future releases of PlaqView will feature more scRNA-seq and scATAC-seq atherosclerosis-related datasets to provide a critical resource for the field, and to promote data harmonization and biological interpretation.